Human embryonic stem cells (hESCs) may provide an unlimited source of insulin-secreting beta-like cells for cell replacement therapy of type 1 diabetes, a disease in which insulin-secreting beta cells in the pancreatic islets of Langerhans are destroyed by autoimmunity. In spite of the recent progress in deriving early definitive and pancreatic endoderm-like progenitors from hESCs, a robust generation of later functional pancreatic beta-like cells has not yet been achieved in vitro. It is increasingly recognized that profound alterations in chromatin structure, including changes in epigenetic DNA methylation and histone modifications, contribute to the control of cell fate decisions. To harness the capability to efficiently differentiate hESCs in vitro, it is necessary to further define epigenetic signatures during pancreatic development. In this Beta Cell Biology Consortium application, we propose to identify and develop novel research resources and tools to advance our long-term goal of enhancing the yield of clinically-applicable human pancreatic beta-like cells from hESCs in culture for the purpose of cell replacement therapy for type 1 diabetes. Specific Aim 1 is to perform epigenome profiling for DNA methylation and key histone marks on two cell populations: mature adult human beta cells, obtained from cadaverous organs, and human ESC-derived pancreatic endocrine like progenitor cells that express neurogenin 3, a transcription factor necessary for the formation of the endocrine pancreas. We will then compare the epigenetic signature of these two populations, which will lead to identification of epigenetic targets and allow development of strategies to manipulate cell differentiation. Specific Aim 2 is to improve the yield of functional beta like cells from hESCs in vitro. We will use three emerging technologies, i.e., macromolecular hydrogels, protein transduction domains, and polyamides, to deliver or inhibit specific molecules that are known, in the literature, to affect beta cell commitment, maturation and function. If successfully completed, the proposed work will be the first data set describing the epigenetic profiles of different human pancreatic cell populations. The novel technologies to be employed will also be valuable for the community to further explore in the field of beta cell biology. Finally, the proposed work will allow us to assess hESC-derived cells in a novel manner, increasing our understanding about whether these cells may be suitable for future clinical applications. PUBLIC HEALTH RELEVANCE: In spite of the recent progress in deriving early definitive and pancreatic endoderm-like progenitors from human embryonic stem cells (hESCs), a robust generation of later functional pancreatic beta-like cells has not yet been achieved in culture. We propose to identify and develop novel research resources (such as epigenetic signatures of beta cells and progenitors) and tools (such as macromolecular hydrogels, protein transduction domains, and polyamides) to advance our long-term goal of enhancing the yield of clinically-applicable human pancreatic beta-like cells from hESCs in culture for the purpose of cell replacement therapy for type 1 diabetes.